Development of Broad-Spectrum Nanobodies for the Therapy and Diagnosis of SARS-CoV-2 and Its Multiple Variants.

The continuous evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has evaded the efficacy of previously developed antibodies and vaccines, thus remaining a significant global public health threat. Therefore, it is imperative to develop additional antibodies that are capable of neutralizing emerging variants. Nanobodies, as the smallest functional single-domain antibodies, exhibit enhanced stability and penetration ability, enabling them to recognize numerous concealed epitopes that are inaccessible to conventional antibodies. Herein, we constructed an immune library based on the immunization of alpaca with the S1 subunit of the SARS-CoV-2 spike protein, from which two nanobodies, Nb1 and Nb2, were selected using phage display technology for further characterization. Both nanobodies, with the binding residues residing within the receptor-binding domain (RBD) region of the spike, exhibited high affinity toward the S1 subunit. Moreover, they displayed cross-neutralizing activity against both wild-type SARS-CoV-2 and 10 ο variants, including BA.1, BA.2, BA.3, BA.5, BA.2.75, BF.7, BQ.1, EG.5.1, XBB.1.5, and JN.1. Molecular modeling and dynamics simulations predicted that both nanobodies interacted with the viral RBD through their complementarity determining region 1 (CDR1) and CDR2. These two nanobodies are novel tools for the development of therapeutic and diagnostic countermeasures targeting SARS-CoV-2 variants and potentially emerging coronaviruses.

Chromosome-level genome assembly and annotation of the Spinibarbus caldwelli.

Spinibarbus caldwelli is an important freshwater economic fish in China. Owing to uncontrolled fishing, wild resources of S. caldwelli have decreased rapidly and may be on the verge of extinction. In this study, utilizing single-molecule real-time (SMRT) sequencing technology and chromatin interaction mapping (Hi-C) technologies, we assembled the first chromosome-scale genome for S. caldwelli about 1.77 Gb in size, with a contig N50 length of 11.83 Mb and scaffold N50 length of 33.91 Mb. In total 1.72 Gb (97.01%) of the contig sequences were anchored onto fifty chromosomes with the longest scaffold being 56.20 Mb. Furthermore, proximately 49.41% of the genome was composed of repetitive elements. In total, 49,377 protein-coding genes were predicted, of which 47,724 (96.65%) genes have been functionally annotated. The high-quality chromosome-level reference genome and annotation are vital for supporting basic genetic studies and will be contribute to genetic structure, functional elucidation, evolutionary inquiry, and germplasm conservation for S. caldwelli.

Comparative genomics analysis and genome assembly integration with the recombination landscape contribute to Takifugu bimaculatus assembly refinement.

Takifugu genus has been brought to the fore in scientific and practical research due to its compact genome, explosive speciation progress and economic value. Here we updated the chromosome-level genome of Takifugu bimaculatus by an ultra-high-density linkage map, a classic and accurate way of chromosome assembly. The map constituted a robust assembly frame, with 92.2% (372.77 Mb) of the draft genome cumulatively placed. With intraspecies and interspecies comparative genomic analysis, we developed a criterion to quantify the differences between assemblies and established a novel way to integrate information from multiple assemblies. The integrated assembly rectified potential mis-assemblies, greatly improving the genome contiguity and correctness. Our results rendered profound information on the genetic recombination of T. bimaculatus and provided new insights into effective genome assembly. The consolidated assembly will be a contributory tool of T. bimaculatus and broadly across the Takifugu by providing a convincing reference for genomic research.

Chromosome-level haplotype-resolved genome assembly for Takifugu ocellatus using PacBio and Hi-C technologies.

Takifugu species serve as a model system for evolutionary studies due to their compact genomes and diverse phenotypes. The ocellated puffer (Takifugu ocellatus), characterized by special colouration, is a scarce anadromous species in the genus Takifugu. As an ornamental and tasty fish species, T. ocellatus has moderate economic value. However, the available genomic resources for this pufferfish are still limited. Here, a chromosome-level reference genome, as well as two haploid genomes, was constructed by PacBio HiFi long sequencing and Hi-C technologies. The total length of the reference genome was 375.62 Mb with a contig N50 of 11.55 Mb. The assembled sequences were anchored to 22 chromosomes with an integration efficiency of 93.78%. Furthermore, 28,808 protein-coding genes were predicted. The haplotype-resolved reference genome of T. ocellatus provides a crucial resource for investigating the explosive speciation of the Takifugu genus, such as elucidating evolutionary histories, determining the genetic basis of trait evolution, and supporting future conservation efforts.

Construction of a High-Density Genetic Linkage Map and QTL Mapping for Growth-Related Traits in Takifugu bimaculatus.

Takifugu bimaculatus is a euryhaline species, distributed ranging from the southern Yellow Sea to the South China Sea. Their tolerance to a wide range of salinity and temperature, coupled with a desirable firm texture, makes T. bimaculatus a strong candidate for Takifugu aquaculture in subtropics areas. Due to the increasing demand in markets and emerging of the Takifugu aquaculture industry, close attention has been paid to improvement on the T. bimaculatus production. In aquaculture, the great effort has been put into marker-assisted selective breeding, and efficient improvement was realized. However, few genetic resources on T. bimaculatus are provided so far. Aiming at understanding the genetic basis underlying important economic growth traits, facilitating genetic improvement and enriching the genetic resource in T. bimaculatus, we constructed the first genetic linkage map for T. bimaculatus via double digestion restriction-site association DNA sequencing and conducted quantitative traits locus (QTL) mapping for growth-related traits. The map comprised 1976 single nucleotide polymorphism markers distributed on 22 linkage groups (LG), with a total genetic distance of 2039.74 cM. Based on the linkage map, a chromosome-level assembly was constructed whereby we carried out comparative genomics analysis, verifying the high accuracy on contigs ordering of the linkage map. On the other hand, 18 QTLs associated with growth traits were detected on LG6, LG7, LG8, LG10, LG20, and LG21 with phenotypical variance ranging from 15.1 to 56.4%. Candidate genes participating in cartilage development, fat accumulation, and other growth-related regulation activities were identified from these QTLs, including col11a1, foxa2, and thrap3. The linkage map provided a solid foundation for chromosomes assembly and refinement. QTLs reported here unraveled the genomic architecture of some growth traits, which will advance the investigation of aquaculture breeding efforts in T. bimaculatus.

Characterization of two myostatin genes in pufferfish Takifugu bimaculatus: sequence, genomic structure, and expression.

Myostatin (MSTN) is a negative regulator of muscle growth, which restrains the proliferation and differentiation of myoblasts. To understand the role of two mstn genes of Takifugu bimaculatus, the full-length cDNAs of 1131 bp Tbmstn1 and 1,080 bp Tbmstn2 were obtained from the T. bimaculatus' genomic database, which encodes 376 and 359 amino acids, respectively. The results of qRT-PCR showed that Tbmstn1 was expressed in the eye, kidney, spleen, skeletal muscle, gill, and brain, and the expression level in the skeletal muscle was extremely significantly higher than in other examined tissues. Tbmstn2 was expressed in the skin, skeletal muscle, gill, and brain, and had the highest expression in the skeletal muscle, followed by expression in the brain. Meanwhile, in different stages of embryonic development, the expression of Tbmstn1 started from the gastrula stage. Its expression in the eye-pigment formation stage and hatching stage was significantly higher than that in other stages. The Tbmstn2 was expressed in all examined embryonic stages with different levels, and the highest expression was detected in the eye-pigment formation stage. These results suggested that Tbmstn1 and Tbmstn2 may involve in the development of skeletal muscle, and Tbmstn2 may be related to the formation of nervous system.

The sequence and de novo assembly of Takifugu bimaculatus genome using PacBio and Hi-C technologies.

Takifugu bimaculatus is a native teleost species of the southeast coast of China where it has been cultivated as an important edible fish in the last decade. Genetic breeding programs, which have been recently initiated for improving the aquaculture performance of T. bimaculatus, urgently require a high-quality reference genome to facilitate genome selection and related genetic studies. To address this need, we produced a chromosome-level reference genome of T. bimaculatus using the PacBio single molecule sequencing technique (SMRT) and High-through chromosome conformation capture (Hi-C) technologies. The genome was assembled into 2,193 contigs with a total length of 404.21 Mb and a contig N50 length of 1.31 Mb. After chromosome-level scaffolding, 22 chromosomes with a total length of 371.68 Mb were constructed. Moreover, a total of 21,117 protein-coding genes and 3,471 ncRNAs were annotated in the reference genome. The highly accurate, chromosome-level reference genome of T. bimaculatus provides an essential genome resource for not only the genome-scale selective breeding of T. bimaculatus but also the exploration of the evolutionary basis of the speciation and local adaptation of the Takifugu genus.